1. Field of the Invention
This invention relates to a low-molecular weight peptide mixture mainly based on dipeptides and tripeptides and a method of producing such a low-molecular weight peptide mixture by incorporating one or more different amino acids covalently into protein by making use of the plastein reaction to thereby form a protein-like material having a modified amino acid composition, and hydrolyzing the protein-like material with a protease to produce a low-molecular with peptide mixture containing peptides having a residue of the amino acid or amino acids incorporated.
2. Description of Prior Art
Traditional nitrogen sources used for nutrition are protein materials, protein hydrolyzates, free amino acid mixtures and the like. It was recently found that low-molecular weight peptides such as di- and tri-peptides play a great role in enteral absorption as described in "Peptide Transport and Hydrolysis" (Elesevier Excepta Media North, Holland, 1977).
The inventors also found that a low-molecular weight peptide mixture mainly based on di- and tri-peptides is a dietetically improved nitrogen source (see Japanese Patent Application No. 55-94169). However, unlike a mixture of free amino acids, a low-molecular weight peptide mixture resulting from hydrolysis of protein material has the disadvantage that its amino acid composition is essentially dependent on that of the starting protein. When a protein material having a limiting amino acid is used or when the amino acid composition should be artificially tailored for the purpose of increasing the content of a particular amino acid, a possible solution is to add a free amino acid to make up the limiting or particular amino acid. Nevertheless, since peptides are enterally absorbed faster than free amino acids and encounter no antagonism upon absorption, it is desirable that such replenishing amino acids are added in the form of peptides.
Making extensive investigations on the production of a low-molecular weight peptide mixture in which the amino acids content of peptide is modified, the inventors have succeeded in producing a desired low-molecular weight peptide mixture by incorporating one or more different amino acids into a protein material through peptide linkages using the plastein reaction and hydrolyzing the resulting modified protein material with a protease.
Although proteolytic enzymes or proteases generally act to decompose peptide linkages, the reaction is rather reversed toward the synthesis of peptide linkages by increasing the substrate concentration. This reverse reaction is called plastein reaction. It is known that when the plastein reaction is used to cause amino acids to act on a protein material, there is obtained a modified protein material in which the amino acids are incorporated in the protein through peptide linkages. When amino acids are incorporated into protein by the reverse reaction of proteolysis, the concentration of protein in a substrate is set to a level from 10% to 60% by weight and higher than in normal hydrolytic reaction and the pH value for the plastein reaction is set more alkaline by pH 2-3 than the optimum pH for the same protease in normal hydrolytic reaction. By carrying out the plastein reaction under such conditions, a modified protein material having particular amino acids covalently incorporated through peptide linkages is produced.
However, it has never been attempted to incorporate at least one amino acid covalently into a protein material through peptide linkages by a simple process.
In incorporating an amino acid covalently into a protein material, the amino acid should be converted into an active form, for example, an ester prior to the reaction because the reaction does not proceed with a free amino acid. A number of methods are known for the esterification of amino acids, including hydrochloric acid, sulfuric acid, and thionyl chloride methods. Improvements are also made in the purification of the resulting amino acid ester. The ester prepared by any of these methods, however, is too expensive for commercial use as long as it must be isolated and purified before use. It is also undesirable in view of safety to the human body to use organic solvents or other chemicals for the purpose of isolating and purifying the amino acid ester. When safety and economy are taken into account, the process is desired to be as simple as possible and use no extra chemicals.